*2.2. Trypanosoma brucei*

Contrary to *Leishmania*, the *T. brucei* life cycle does not require the intracellular environment for any of its developmental forms. *T. brucei* is transmitted between mammalian hosts by *Glossina* spp. (tsetse fly), in which the bloodstream short stumpy form (B-SS) differentiates into the replicative procyclic form (PFs). PFs migrate to the proventriculus were they subsequently differentiate into epimastigotes and into cycle-arrested metacyclics (infective form) in the salivary glands of the tsetse fly. Parasites colonize the mammalian host during the blood meal of the fly and differentiate into bloodstream long slender form (B-LS), which eventually evolves to the B-SS form by a quorum-sensing mechanism [48,49]. Consequently, drug-screening assays targeting *T. brucei* rely on the bloodstream form of the parasite. Different approaches for whole-cell, high-throughput screening have recently been successfully developed. Mackey et al. (2006) screened 2160 FDA-approved drugs, bioactive compounds, and natural products to identify hits that were cytotoxic to *T. brucei* at a concentration of 1 µM or less. This approach led to the identification of 35 new hits from seven different drug categories, which included two approved trypanocidal drugs, suramin and pentamidine [50]. Similar to *Leishmania*, bioluminescent-engineered *T. brucei* have recently been developed and implemented in whole-cell high-throughput screens. Sykes et al. (2009) developed a luciferase-based viability assay for ATP detection in a 384-well format, making high-throughput whole-cell screening in *T. brucei* very reproducible, sensitive, and cost effective [51]. Later, Sykes et al. (2012) described the application of an

Alamar Blue (resazurin)-based, 384-well high-throughput screening (HTS) assay to screen a library of 87,296 compounds, leading to 6 hits from 5 new chemical classes displaying great activity against *T.b. rhodesiense* [52]. As an alternative to luciferase and Alamar Blue, Faria et al. (2015) developed a whole-cell assay in 384-well plates based on the quantitative detection of double-stranded DNA bound to cyanine dye SYBR Green. The assay was a validated screening of a kinase-focused library composed of 4000 compounds, leading to the discovery of novel scaffolds with potent antitrypanosomal activity [53]. In the recent years, thanks to different screening initiatives, several new leads such as diamidine derivatives, fexinidazole, oxaborole SCYX-7158, quinolone amide GHQ168, and acoziborole are now in various stages of the development pipeline for treating HAT [54–56].

#### *2.3. Trypanosoma cruzi*

Infective trypomastigotes and intracellular replicative amastigotes are the clinically relevant life-cycle stages of *T. cruzi* that are targets for drug intervention [57]. Briefly, non-dividing *T. cruzi* metacyclic trypomastigotes are transmitted to humans in the feces of infected triatomine bugs at the bite site of these hematophagous insects. Trypomastigotes invade various cell types and transform into intracellular amastigotes, which multiply by binary fission until the host cell is overwhelmed, and then transform into bloodstream trypomastigotes and spread to distant sites through the lymphatics and bloodstream. Once back in the insect vector, trypomastigotes transform into epimastigotes and then differentiate into infective metacyclic trypomastigotes [58]. Despite many efforts, only two compounds, benznidazole (since 1972) and nifurtimox (since 1967), are currently used for the treatment of certain forms of Chagas disease [59]. Markedly, drug discovery in *T. cruzi* is handicapped by the small number of well-established targets (e.g., the sterol biosynthetic pathway, cruzipain, cytochrome b, trypanothione reductase, cyclophilin, or carbonic anhydrases [57]), which explains the wide use of phenotypic approaches that have become the main pillar of Chagas R&D [60]. Drug screening against *T. cruzi* can be performed in cell-free axenic amastigotes and epimastigotes, as well as in intracellular amastigotes, with similar advantages and caveats to those previously discussed for *Leishmania*. In terms of tools for measuring the trypanocidal effect of the compounds, screening systems have evolved from manual microscopic counting of parasite growth; the use of colorimetric substrates (e.g., chlorophenol-red-β-D-galactopyranoside); bioluminescent (e.g., parasites expressing the firefly luciferase) and fluorescent reporters (e.g., tdTomato-expressing lines); and high-content imaging approaches that do not require the incorporation of any reporter molecule [35,61,62]. Engel et al. (2010) developed a cell-based HTS assay that can be used with untransfected *T. cruzi* isolates and host cells that can simultaneously measure efficacy against the parasite and host cell toxicity. This approach was used to screen a library of 909 bioactive compounds, leading to the identification of 55 hits [63]. Using NIH-3T3 fibroblasts infected with a recombinant *T. cruzi* strain expressing beta-galactosidase as an intracellular reporter, Peña et al. (2015) screened the GlaxoSmithKline diversity set of 1.8 million compounds. A total of 2310 compounds were identified with great potency against *T. cruzi* (pIC<sup>50</sup> > 5) and a selectivity index > 10 [64]. The resulting lead compounds were further validated by Alonso-Padilla et al. (2015) using a novel, highly reproducible, high-content, high-throughput assay using myoblasts [65]. De Rycker et al. (2016) developed a new hit discovery screening cascade designed combining a primary imaging-based assay followed by newly developed and appropriately scaled secondary assays to predict the cidality and rate-of-kill of the compounds. This cascade was used to profile the SelleckChem set (421 FDA-approved drugs) and the NIH Clinical Collection set (727 compounds that have been used in clinical trials), leading to the identification of several known clinical compounds as candidates for a repurposing strategy for Chagas disease [66]. This cascade was further improved by the inclusion of three distinct in vitro assays: the slow replicating/cycling strain potency assay, the trypomastigote assay, and the extended duration washout assay [67]. Recently, Bernatchez et al. (2020) screened 7680 compounds from the Repurposing, Focused Rescue, and Accelerated Medchem library, and identified seven lead compounds with potent in vitro activity against *T. cruzi* and good therapeutic index [68].
